Method for the production of D-pantothenic acid and/or salts thereof via purification by nanofiltration as adjunct for animal feedstuffs

ABSTRACT

The invention relates to an improved method for the production of D-pantothenic acid and/or salts thereof and use thereof as adjunct for animal feedstuffs.

This application is the U.S. national phase of International ApplicationNo. PCT/EP02/01754, filed on Feb. 20, 2002, which claims priority toGerman Application No. 10108226.6, filed on Feb. 21, 2001.

NAMES OF PARTIES TO A JOINT RESEARCH AGREEMENT

Subject matter described and claimed in the instant patent was developedpursuant to a joint research agreement between OmniGene Bioproducts,Inc. and BASF Aktiengesellschaft.

The present invention relates to an improved process for preparingD-pantothenic acid and/or salts thereof and the use as additive toanimal feedstuffs.

TECHNICAL FIELD

D-pantothenate is widespread in the plant and animal kingdom as astarting product for the biosynthesis of coenzyme A. In contrast tohumans, who consume pantothenic acid in sufficient quantities via thediet, symptoms of D-pantothenate deficiency are, however, frequentlydescribed not only for plants but also for animals. The availability ofD-pantothenate is therefore of great economic interest, in particular inthe animal feed industry.

Conventionally, D-pantothenate is prepared by chemical synthesis fromD-pantolactone and calcium β-alaninate (Ullmann's Encyclopedia ofIndustrial Chemical, 6th edition, 1999, electronic release, Chapter“Vitamins”). To prepare D-pantolactone, a complex classical racemateseparation via diastereomeric salts is required. The commercial productresulting from the chemical synthesis is usually the calcium salt ofD-pantothenic acid, calcium D-pantothenate.

Compared with chemical synthesis, the advantage of biotechnologicalpreparation processes using microorganisms is in the selective(enantiomerically pure) provision of the D form of pantothenic acidwhich can be utilized by higher organisms. A complex racemate separationas is required in chemical synthesis is thus unnecessary.

BACKGROUND ART

Fermentation processes for preparing D-pantothenic acid bymicroorganisms have been disclosed in great number, including by EP 0590 857, WO 96/33283; U.S. Pat. No. 6,013,492, WO 97/10340, DE 198 46499, EP 1 001 027, EP 1 006 189, EP 1 006 192 and EP 1 006 193.

Thus EP 1 006 189 and EP 1 001 027 describe processes for preparingpantothenate in which the fermentation solution reaches a content of atmost 1 g/l of D-pantothenic acid. However, such low pantothenic acidcontents in the fermentation solution, that is to say less than 10% byweight based on the solids content, are unsuitable for economicproduction of D-pantothenic acid-containing animal feed supplements. Afurther disadvantage of the processes described to date is thatisolating the product from the fermentation medium requires numerouscomplex work-up steps. An economic production process for the industrialscale has not been disclosed.

In the German laid-open application DE 100 16 321, a fermentationprocess is described for producing a D-pantothenic-acid-containinganimal feed supplement. However, a significant disadvantage of thisprocess, as also with the fermentation processes cited above forD-pantothenic acid production, is that the pantothenic acid precursorβ-alanine has to be supplied to the microorganism via the fermentationmedium in order to obtain economic yields of the desired product.

In addition U.S. Pat. No. 6,013,492 and WO 96/332839 describe thework-up of D-pantothenic acid from the fermentation solution byfiltering off insoluble constituents (e.g. cell material) from theculture medium, adsorbing the filtrate to activated carbon, subsequentlyeluting the D-pantothenic acid with an organic solvent, preferablymethanol, neutralizing the eluent with calcium hydroxide, and finallycrystallizing calcium D-pantothenate. Significant disadvantages are thelosses of product of value which occur during the crystallization andthe use of an organic solvent which may be removed from the product onlywith difficulty and requires complex solvent recovery.

EP 0 590 857 describes a fermentation process for producingD-pantothenic acid in which culturing a microorganism obligatorilyrequires supply of β-alanine. The fermentation solution is filtered toseparate off the biomass, then passed through a cationic exchanger andthen through an anionic exchanger, is then neutralized with calciumhydroxide, concentrated by evaporation, admixed with activated carbon,filtered once more and crystallized with addition of methanol andcalcium chloride. The resultant calcium-pantothenate-containing product,in addition to D-pantothenic acid in the form of the calcium salt, alsocontains calcium chloride in a molar ratio of 1:1. To reduce the calciumchloride content, electrodialysis with subsequent spray-drying isnecessary. This process has the disadvantage, because of themultiplicity of complex process steps and the use of organic solvents,of being neither economic nor ecological.

DISCLOSURE OF INVENTION

It is an object of the present invention to provide an animal feedsupplement containing D-pantothenic acid and/or salts thereof and alsothe production thereof by an improved process for preparingD-pantothenic acid and/or salts thereof which does not have thedisadvantages mentioned above. In this case, for economic reasons, aprocess is desirable in which supply of β-alanine is drastically reducedor is not required at all. In addition, it is desirable to prepareD-pantothenic acid in the form of its divalent salts and, in this caseespecially the alkaline earth metal salts, since the divalent salts havelower hygroscopic properties than monovalent salts of pantothenic acidand for further use, for example as animal feed supplement, thus have aless pronounced tendency toward aggregation.

We have found that this object is achieved in an advantageous manner bythe present invention.

The present invention relates to a process for preparing D-pantothenicacid and/or salts thereof, which comprises

-   a) using at least one D-pantothenic-acid-producing organism, the    pantothenic acid (pan) and/or isoleucine/valine (ilv) biosynthesis    of which is deregulated and which forms at least 2 g/l of salts of    D-pantothenic acid by fermentation in a culture medium, with 0-20    g/l of free β-alanine and/or β-alanine salt being fed to the culture    medium,-   b) salts containing polyvalent cations being fed to the    D-pantothenate formed, polyvalent salts of D-pantothenic acid being    formed,-   c) the solution containing polyvalent salts of D-pantothenic acid    being worked up by nanofiltration, the polyvalent salts of    D-pantothenic acid being enriched and-   d) the nanofiltration retentate containing polyvalent salts of    D-pantothenic acid being subjected to drying and/or formulation.

MODE(S) FOR CARRYING OUT THE INVENTION

In a variant of the inventive process, the retentate from step c) is asuspension containing polyvalent salts of D-pantothenic acid.

In addition, the fermentation can be carried out by procedures known perse in the batch, fed-batch or repeated fed-batch mode or undercontinuous process procedures. For neutralizing the resultingpantothenic acid, customary buffer systems are used in this case, forexample phosphate buffer containing NaOH, KOH or ammonia.

In further variants of the inventive process, in step a), at least 10g/l, preferably at least 20 g/l, particularly preferably at least 40g/l, very particularly preferably at least 60 g/l, and in particular atleast 70 g/l of salts of D-pantothenic acid are formed in the culturemedium by fermentation.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of the present invention, the formulation “produce”means that the organism can synthesize greater amounts of D-pantothenicacid and/or salts thereof than are required for its own metabolic needs.In an inventively advantageous variant, the amount of D-pantothenic acidand/or salts thereof which is synthesized is not present in the cellinterior, but, ideally, is completely released into the culture mediumfrom the organism. This secretion can take place actively or passivelyby mechanisms which are known per se.

According to the invention, the D-pantothenic-acid-producing organismsused are microorganisms. These include, according to the invention,fungi, yeasts and/or bacteria. According to the invention preference isgiven to fungi, for example Mucor, or yeasts, for example Saccharomycesor Debaromyces [sic] and, in this case, preference is given to usingSaccharaomyces [sic] cerevisiae. Advantageously according to theinvention coryneform bacteria or Bacillaceae are used. Those which arecovered according to the invention are preferably, for example, bacteriaof the genera Corynebacterium, Escherichia, Bacillus, Arthrobacter,Bevibacterium, Pseudomonas, Salmonella, Klebsiella, Proteus,Acinetobacter or Rhizobium. Particular preference is given here, forexample, to Corynebacterium glutamicum, Brevibacterium breve orBaccillus subtilis, B. licheniformis, B. amyloliquefaciens, B. cereus,B. lentimorbus, B. lentus, B. firmus, B. pantothenticus, B. circulans,B. coagulans, B. megaterium, B. pumilus, B. thuringiensis, B. brevis, B.stearothermophilus and other bacillus species of group 1 which arecharacterized by their 16sRNA, or Actinum mycetalis. This listing servesfor illustration and is in no way limiting for the present invention.

Furthermore, the present invention also comprises the use of geneticallymodified organisms for the inventive preparation of an animal feedsupplement containing free D-pantothenic acid and/or salts thereof. Suchgenetically modified organism can be isolated, for example, by chemicalmutagenesis and subsequent selection by a suitable “screening method”.The invention also comprises what are termed “production strains”, whichare suitable for producing the product within the meaning of the presentinvention and have genetic modifications with respect to metabolic fluxin the direction of the D-pantothenic acid, other modifications withrespect to the secretion of D-pantothenic acid and/or salts thereofthrough the cell membrane also being included. This can be achieved, forexample, by modifications at key positions in relevant metabolicbiosynthesis pathways of the organism used.

It is also conceivable to use transgenic organisms which result from thetransfer of homologous and/or heterologous nucleotide sequences whichare required or can be beneficial for the synthesis of the desiredproduct. In this case the overexpression and/or deregulation of one ofmore genes individually and/or in combination, localized in the genomeand/or on a vector is conceivable.

Such transgenic organisms can contain, advantageously, additional copiesof and/or genetically modified genes selected from the group consistingof panB, panC, panD, panE and/or combinations thereof and/or evenorganizational units, such as the panBCD operon. In addition, furthermetabolic pathways, for example the isoleucine-valine biosyntheticpathway can be advantageously manipulated in the organisms, for exampleas described in EP 1 006 189, EP 1 006 192, EP 1 006 193 or EP 1 001027. By this means, branched-chain precursor substances of pantothenicacid biosynthesis are provided in greater quantity. Advantageously, ifappropriate, the genes for this biosynthetic pathway, that is to sayilvB, ilvN, ilvC and/or ilvD, are overexpressed.

Furthermore, genetic modifications of aspartate-α-decarboxylase (panD),for example by overexpression and/or deregulation, are comprisedaccording to the invention in the D-pantothenic-acid-producing organismused.

For the purposes of the present invention, the formulation“deregulation” means the following: change or modification of at leastone gene which codes for an enzyme in a biosynthetic metabolic pathway,so that the activity of the enzyme in the microorganism is changed ormodified. Preferably, at least one gene which codes for an enzyme of abiosynthetic metabolic pathway is altered in such a manner that the geneproduct is formed to an increased extent or has an increased activity.The term “deregulated metabolic pathway” also includes a biosyntheticmetabolic pathway in which more than one gene which codes for more thanone enzyme is changed or modified in such a manner that the activitiesof more than one enzyme are changed or modified.

Changes or modifications can comprise, but are not limited to: removingthe endogenous promoter or regulatory elements; introducing strongpromoters, inducible promoters or a plurarity of promoterssimultaneously; removing regulatory sequences, so that the expression ofthe gene product is changed; changing the chromosomal position of thegene; changing the DNA sequence in the vicinity of the gene or withinthe gene, for example of the ribosomal binding site (RBS); increasingthe number of copies of the gene in the genome or by introducingplasmids of differing number of copies; modification of proteins (e.g.regulatory proteins, suppressors, enhancers, transcriptional activatorsand the like) which play a role in the transcription of the gene and/orin the translation to give the gene product. These also include allother possibilities for deregulating the expression of genes which areprior art, for example the use of antisense oligonucleotides, or theblocking of repressor proteins.

Deregulation can also comprise changes in the coding region of geneswhich lead, for example, to removing feedback regulation in the geneproduct or to a higher or lower specific activity of the gene product.

Furthermore, according to the invention genetic engineering changes toenzymes are advantageous which effect the efflux of precursors ofpantothenic acid and/or the flux of pantothenic acid to coenzyme A.Examples of such enzyme-coding genes are: alsD, avtA, ilvE, ansB, coaA,coaX etc. This listing serves for illustration and is in no way limitingfor the present invention.

In addition, genetic engineering changes are advantageous which securethe cellular provision of cofactors (for example of methylenetetrahydrofolate, redox equivalents etc.) in an amount optimum forpantothenic acid production.

Advantageously, β-alanine is thus already present in the cells atincreased concentrations compared with correspondingly non-geneticallymodified organisms, and thus need not be added as a precursor to theculture medium, as is required, for example, in EP-A-0 590 857.Microorganisms are advantageous in which pantothenic acid (pan) and/orisoleucine/valine (ilv) biosynthesis and/or asparate [sic]α-decarboxylase (panD) is deregulated. Furthermore, an additionaloverexpression of ketopanthoate reductase (panE) in the microorganismsis advantageous.

It is further advantageous according to the invention if the coaA gene,which is required for the synthesis of coenzyme A, if appropriate isreduced in activity or (for example in Bacillus species) is entirelyswitched off. This is because Bacillus, in addition to coaA, contains afurther gene for this enzymatic function (=coaX). The activity of thiscoaX gene or of the corresponding enzyme can also be altered, preferablyreduced, or even deleted, provided that coaA itself still hassufficient, even if decreased, enzyme activity, that is to say theenzyme activity of coaA has not entirely been lost. In addition tooverexpressing the various genes, genetic manipulation of the promoterregions of these genes is advantageous in such a manner that thismanipulation leads to an overexpression of the gene products.

In a variant embodiment of the present invention, the bacterial strainsdescribed according to the annex (PCT/US application 0025993), forexample Bacillus subtilis PA824 and/or derivatives thereof are used. Ina further variant embodiment, according to the invention themicroorganism Bacillus subtilis PA668, as described according to theannex (U.S. serial No. 60/262,995) is used in the inventive process.These strains Bacillus subtilis PA824 and PA668 were produced asfollows:

Starting from the strain Bacillus subtilis 168 (Marburg strain ATCC6051), which has the genotype trpC2 (Trp⁻), the strain PY79 was producedvia transduction of the Trp⁺ marker (from the Bacillus subtilis wildtype W23). ΔpanB and ΔpanE1 mutations were introduced into strain PY79by classical genetic engineering methods (as described, for example, inHarwood, C. R. and Cutting, S. M. (editors), Molecular BiologicalMethods for Bacillus (1990) John Wiley & Sons, Ltd.; Chichester,England).

The resultant strain was transformed using genomic DNA of Bacillussubtilis strain PA221 (genotype P₂₆panBCD, trpC2 (Trp⁻)) and genomic DNAfrom Bacillus subtilis strain PA303 (genotype P₂₆panE1). The resultantstrain PA327 has the genotype P₂₆panBCD, P₂₆panE1 and is a typotophanauxotroph (Trp⁻). Using the Bacillus subtilis strain PA327, in 10 mlcultures using SVY medium (25 g/l of Difco Veal Infusion Broth, 5 g/l ofDifco Yeast Extract, 5 g/l of Na glutamate, 2.7 g/l of ammonium sulfate,make up to 740 ml with water, autoclave, then add 200 ml of 1 Mpotassium phosphate, pH 7.0 and 60 ml of 50% sterile glucose solution),which had been supplemented with 5 g/l of β-alanine and 5 g/l ofα-ketoisovalerate, pantothenic acid titers of up to 3.0 g/l (24 h) was[sic] achieved.

The preparation of Bacillus subtilis strain PA221 (genotype P₂₆panBCD,trpC2 (Trp⁻)) is described in the following section:

By classical genetic engineering methods, with the aid of the sequenceinformation of the panBCD operon of E. coli (see Merkel et al., FEMSMicrobiol. Lett., 143 1996:247-252), starting from a Bacillus subtilisGP275 plasmid library, the panBCD operon of Bacillus was cloned. For thecloning, the E. coli strain BM4062 (bir^(ts)) was used, as was theinformation that the Bacillus operon is in the vicinity of birA gene.The panBCD operon was introduced into a plasmid which can replicate inE. coli. To improve the expression of the panBCD operon, strongconstitutive promoters of Bacillus subtilis phages SP01 (P₂₆) were usedand the ribosome binding site (═RBS) in front of the panB gene wasreplaced by an artificial RBS. A DNA fragment which is immediatelyupstream of the native panB gene in Bacillus, was ligated in front ofthe P₂₆panBCD cassette on the plasmid. This plasmid was transformed intothe Bacillus subtilis strain RL-1 (derivative of Bacillus subtilis 168obtained by classical mutagenesis (Marburg strain ATCC 6051), genotypetrpC2 (Trp⁻)) and, by homologous recombination, the native panBCD operonwas replaced by the p₂₆panBCD operon. The resultant strain is termedPA221 and has the genotype P₂₆panBCD, trpC2 (Trp⁻).

Using the Bacillus subtilis strain PA221, in 10 ml cultures with SVYmedium which have been supplemented with 5 g/l of β-alanine and 5 g/l ofα-ketoisovalerate, pantothenic acid titers of up to 0.92 g/l (24 h) was[sic] achieved.

Production of the Bacillus subtilis strain PA303 (genotype P₂₆panE1) isdescribed in the following section:

With the aid of the E. coli pane gene sequence, the Bacillus panEsequence was similarly cloned. It was found that two homologs of thepanE gene of E. coli exist in B. subtilis which are termed panE1 andpanE2. By deletion analysis, it was found that the panE1 gene isresponsible for 90% of pantothenic acid production, while deleting thepanE2 gene had no significant effect on the pantothenic acid production.Here also, in a similar manner to cloning the panBCD operon, thepromoter was replaced by the strong constitutive promoter P₂₆ and theribosome binding site before the panE1 gene was replaced by theartificial binding site. The P₂₆panE1 fragment was cloned into a vectorwhich was designed so that the P₂₆panE1 fragment could integrate intothe original panE1 locus in the genome of Bacillus subtilis. The strainresulting after transformation and homologous recombination is termedPA303 and has the genotype P₂₆panE1.

Using the Bacillus subtilis strain PA303, in 10 ml cultures using SVYmedium which had been supplemented with 5 g/l of β-alanine and 5 g/l ofα-ketoisovalerate, pantothenic acid titers of up to 1.66 g/l (24 h) was[sic] achieved.

Further strain construction proceeded via transformation of PA327 usinga plasmid which contains the P₂₆ilvBNC operon and the marker gene forspectinomycin. The P₂₆ilvBNC operon integrated into the amyE locus,which was demonstrated by PCR. One transformant was termed PA340(genotype P₂₆panBCD, P₂₆panE1, P₂₆ilvBNC, specR, trpC2 (Trp⁻)).

Using the Bacillus subtilis strain PA340, in 10 ml cultures using SYVmedium which had only been supplemented with 5 g/l of β-alanine,pantothenic acid titers of up to 3.6 g/l (24 h) was [sic] achieved, andin 10 ml cultures using SVY medium which had been supplemented with 5g/l of β-alanine and 5 g/l of α-ketoisovalerate, pantothenic acid titersof up 4.1 g/l (24 h) were achieved.

In addition, a deregulated ilvD cassette was introduced into strainPA340. For this, a plasmid containing the ilvD gene under the control ofthe P₂₆ promoter having the artificial RBS2 was transformed into PA340.In this case the P₂₆ilvD gene was integrated into the original ilvDlocus by homologous recombination. The resultant strain PA374 has thegenotype P₂₆panBCD, P₂₆panE1, P₂₆ilvBNC, P₂₆ilvD, specR and trpC2(Trp⁻).

Using the Bacillus subtilis strain PA374, in 10 ml cultures using SVYmedium which was supplemented only with 5 g/l of β-alanine, pantothenicacid titers of up to 2.99 g/l (24 h) was [sic] achieved.

To produce pantothenic acid using strain PA374 without supplyingβ-alanine, additional copies of the gene panD coding for aspartateα-decarboxylase were introduced into strain PA374. For this, chromosomalDNA of strain PA401 was transformed into PA374. The strain PA377 wasobtained by selection on tetracycline.

The resultant strain PA377 has the genotype P₂₆panBCD, P₂₆panE1,P₂₆ilvBNC, P₂₆ilvD, specR, tetR and trpC2 (Trp⁻).

Using the Bacillus subtilis strain PA377, in 10 ml cultures using SVYmedium, precursor-supply-free pantothenic acid titers of up to 1.3 g/l(24 h) was [sic] achieved.

The preparation of the Bacillus subtilis strain PA401 (genotype P₂₆panD)is described in the following section:

The Bacillus subtilis panD gene was cloned from the panBCD operon into avector bearing the tetracyclin marker gene. The promoter P₂₆ and anabove-described artificial RBS were cloned in front of the panD gene. Byrestriction digestion, a fragment containing the tetracyclin marker geneand the P₂₆panD gene was produced. This fragment was religated andtransformed into the above-described strain PA221. The fragmentintegrated into the genome of strain PA211. The resultant strain PA401has the genotype P₂₆panBCD, P₂₆panD, tetR and trpC2 (Trp⁻).

Using Bacillus subtilis strain PA401, in 10 ml cultures in SVY mediumwhich had been supplemented with 5 g/l of α-ketoisovalerate, pantothenicacid titers of up to 0.3 g/l (24 h) was [sic] achieved. In 10 mlcultures using SVY medium which had been supplemented with 5 g/l ofD-pantoin acid and 10 g/l of L-aspartate, pantothenic acid titers of upto 2.2 g/l (24 h) were achieved.

Starting from strain PA377, by transformation with chromosomal DNA fromstrain PY79, a tryptophan prototroph strain was generated. This strainPA824 has the genotype P₂₆panBCD, P₂₆panE1, P₂₆ilvBNC, P₂₆ilvD, specR,tetR and Trp⁺.

Using Bacillus subtilis strain PA824, in 10 ml cultures in SVY medium,precursor-supply-free pantothenic acid titers of up to 4.9 g/l (48 h)was [sic] achieved (control PA377: up to 3.6 g/l in 48 h). The exactconstruction of the strains is as given according to the annex of PCT/USapplication 0025993.

The preparation of PA668 is described in the following section:

The Bacillus panB gene was cloned from the wild type panBCD operon andinserted into a vector which, in addition to a chloramphenicolresistance gene, also contains B. subtilis sequences of the vpr locus.

The strong constitutive promoter P₂₆ was introduced in front of the 5′end of the panB gene. A fragment which contains the P₂₆panB gene, themarker gene for chloramphenicol resistance and Bacillus subtilis vprsequences was obtained by restriction digestion. The isolated fragmentwas religated and the strain PA824 was transformed therewith. Theresultant strain was termed PA668. The genotype of PA668 is: P₂₆panBCD,P₂₆panE1, P₂₆ilvBNC, P₂₆ilvD, P₂₆panB, specR, tetR, CmR and Trp⁺.

Two colonies of PA668 were isolated and termed PA668-2A, and the otherPA668-24.

Using B. subtilis strain PA668-2A, in 10 ml cultures in SVY mediumwithout supply of precursors, pantothenic acid titers of 1.5 g/l areachieved in 48 h. In 10 ml cultures which are supplemented with 10 g/lof aspartate, titers up to 5 g/l are achieved. Using B. subtilis strainPA668-24, in 10 ml cultures in SVY medium without supplying precursors,pantothenic acid titers of 1.8 g/l are achieved in 48 h. In 10 mlcultures supplemented with 10 g/l of L-aspartate, titers up to 4.9 g/lare achieved.

The exact construction of the strains is given according to the annexesof PCT/US application 0025993 and U.S. serial No. 60/262,995.

Using the above-described strain PA377, in glucose-limited fermentationin SVY medium (25 g/l of Difco Veal Infusion Broth, 5 g/l of Difco YeastExtract, 5 g/l of tryptophan, 5 g/l of Na glutamate, 2 g/l of (NH₄)₂SO₄,10 g/l of KH₂PO₄, 20 g/l of K₂HPO₄, 0.1 g/l of CaCl₂, 1 g/l of MgSO₄, 1g/l of sodium citrate, 0.01 g/l of FeSO₄.7H₂O and 1 ml/l of a trace saltsolution of the following composition: 0.15 g of Na₂MoO₄.2H₂O, 2.5 g ofH₃BO₃, 0.7 g of CoCl₂.6H₂O, 0.25 g of CuSO₄.5H₂₀, 1.6 g of MnCl₂.4H₂O,0.3 g of ZnSO₄.7H₂O made up to 1 l with water)) on a 10 l scale withcontinuous supply of a glucose solution, pantothenic acid concentrationsof 18-19 g/l (22-25 g/l) in the fermentation broth are achieved in 36 h(48 h).

In glucose-limited fermentation of PA824, the tryptophan-prototrophderivative of PA377, in yeast extract medium (10 g/l of Difco YeastExtract, 5 g/l of Na glutamate, 8 g/l of (NH₄)₂SO₄, 10 g/l of KH₂PO₄, 20g/l of K₂HPO₄, 0.1 g/l of CaCl₂, 1 g/l of MgSO₄, 1 g/l of sodiumcitrate, 0.01 g/l of FeSO₄.7H₂O and 1 ml/l of the above-described tracesalt solution) on a 10 l scale with continuous supply of a glucosesolution, the following pantothenic acid concentrations are achieved infermentation broths in 36 h, 48 h and 72 h: 20 g/l, 28 g/l and 36 g/l.

By further media optimization, using strain PA824 in glucose-limitedfermentation in a medium consisting of 10 g/l of Difco Yeast Extract, 10g/l of NZ amine A (Quest International GmbH, Erftstadt), 10 g/l of Naglutamate, 4 g/l of (NH₄)₂SO₄, 10 g/l of KH₂PO₄, 20 g/l of K₂HPO₄, 0.1g/l of CaCl₂, 1 g/l of MgSO_(4, 1) g/l of sodium citrate, 0.01 g/l ofFeSO₄.7H₂O and 1 ml/l of the above-described trace salt solution on a 10l scale with continuous supply of a glucose solution, pantothenic acidconcentrations of 37 g/l (48 g/l) is [sic] achieved in fermentationbroths in 36 h (48 h).

Further increases in pantothenic acid concentration in the fermentationbroth are conceivable by further media optimization, by increasing thefermentation time, by process and strain improvement and by combinationsof the individual steps. Thus the above-described pantothenic acidconcentrations may also be achieved by fermentation of strains which arederivatives of the above-described PA824. Derivatives can be produced byclassical strain development and by further genetic engineeringmanipulations. By development of media, strain and fermentationprocesses, the pantothenic acid titers in the fermentation broths can beincreased to greater than 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 and >90g/l.

An important advantage of the inventive process is that the fermentationis carried out in a culture medium which, apart from at least one carbonand nitrogen source as starting compounds does not contain furtherprecursors. That is to say the biosynthesis of D-pantothenic acid isindependent of the supply of further precursors. For the purposes of thepresent invention such precursors are, for example, β-alanine and/orL-aspartate and/or L-valine and/or α-ketoisovalerate and/or combinationsthereof.

In a preferred variant of the inventive process, the fermentation of theD-pantothenic-acid-producing organism is carried out in a culture mediumwhich contains a carbon source and a nitrogen source, to which, however,no free β-alanine and/or β-alanine salts are added or is carried out inthe course of the fermentation. That is to say, to produce D-pantothenicacid in ranges of at least 10 g/l of culture medium, preferably at least20 g/l, particularly preferably at least 40 g/l, very particularlypreferably at least 60 g/l, and in particular at least 70 g/l, accordingto the invention no supply of free β-alanine and/or β-alanine salts isnecessary.

The independence of the supply of precursors is, in particular, animportant economic advantage of the inventive process compared withknown processes, since a multiplicity of precursors are very expensive.

However, adding β-alanine and/or β-alanine salts is not excludedaccording to the invention, so that therefore the yield of D-pantothenicacid can be further improved by adding β-alanine and/or β-alanine salts.Assuming, for example, that all required precursors of pantothenic acidare present in a sufficient amount, only the activity of the panD genelimits a further increase in pantothenic acid production, then the yieldof pantothenic acid can be increased, for example, by a further 50% byadding free β-alanine and/or β-alanine salts.

In an advantageous variant of the present invention, up to 20 g/l offree β-alanine and/or β-alanine salts can be added to the culture mediumfor additional increase in pantothenic acid yield by more than 50%.Preference is given to adding about 15 g/l of free β-alanine and/orβ-alanine salts to the culture medium.

Examples of carbon sources suitable according to the invention for usein a culture medium for fermentation of the abovementioned organisms aresugars, such as starch hydrolyzates (mono-, di-, oligosaccharides),preferably glucose or sucrose, and also beet or cane sugar molasses,proteins, protein hydrolyzates, soybean flour, corn steep liquor, fats,free fatty acids, recirculated cells from fermentations carried outpreviously or hydrolyzates thereof and also yeast extract. Theselistings are not limiting for the present invention.

In addition, the present invention is advantageously distinguished inthat the total sugar content is reduced to a minimum toward the end ofthe fermentation, since otherwise this makes difficult the later dryingand/or formulation of the fermentation solution due to sticking. Thiscan be achieved according to the invention by carrying on thefermentation for some further time after the carbon source is exhausted(in the case of batch culture) or after the carbon feed (in the case offed-batch or repeated fed-batch process conditions) is interruptedand/or regulated in such a manner that the concentration of the carbonsource is virtually zero (in the case of fed-batch, repeated fed-batchor continuous process conditions).

This is carried out according to the invention by carrying on thefermentation, after interruption in addition of the carbon source (forexample sugar solution), until the dissolved oxygen concentration (pO₂)reaches at least 80%, preferably 90% and particularly preferably 95%, ofthe saturation value in the fermentation solution.

Examples of nitrogen sources suitable according to the invention areammonia, ammonium sulfate, urea, proteins, protein hydrolyzates or yeastextract. This listing is also not limiting for the present invention.

In addition, the fermentation medium contains mineral salts and/or traceelements, such as amino acids and vitamins. The exact compositions ofsuitable fermentation media are known in large numbers and accessible tothose skilled in the art.

After inoculating the fermentation medium with a suitableD-pantothenic-acid-producing organism (at cell densities known to thoseskilled in the art), if appropriate with addition of an antifoam, theorganism is cultured. Any necessary regulation of the medium pH can becarried out using various inorganic or organic alkali metal hydroxidesolutions or acids, for example NaOH, KOH, ammonia, phosphoric acid,sulfuric acid, hydrochloric acid, formic acid, succinic acid, citricacid or the like.

Owing to the buffer systems used during the fermentation, which can beas previously described, for example NaOH, KOH, ammonia, phosphoricacid, sulfuric acid, hydrochloric acid, formic acid, succinic acid,citric acid or the like, the D-pantothenic acid formed in thefermentation solution, depending on the buffer system used, is presentin the form of the respective salt(s). Since in this case, inparticular, the salts of D-pantothenic acid in the form of theirmonovalent cations are disadvantageous, the fermentation solution isworked up according to the invention by nanofiltration.

For this, salts containing polyvalent cations are first fed according tothe invention to the D-pantothenate formed, which forms polyvalent saltsof D-pantothenic acid. According to the invention, salts containingpolyvalent cations can be added in solid form or in aqueous solutionduring, preferably at the end of, or after, the fermentation in step a).An aqueous solution containing polyvalent cations can be fed, forexample, continuously.

In addition, in a step upstream of the nanofiltration, that is to saybefore the nanofiltration in step c) of the inventive process, cell massor components precipitated in the solution can be separated off. In thiscase the separation can be performed by decanting or membranefiltration, preferably ultrafiltration. In a variant of the inventiveprocess, the membrane filtration is carried out as diafiltration. Herealso, according to the invention salts containing polyvalent cations canbe added in solid form or aqueous solution during, or after, a membranefiltration of a D-pantothenate-containing solution. For example, anaqueous solution containing polyvalent cations is fed here continuously.

In the separation of cell mass and/or components precipitated out in thesolution, for example slightly soluble or insoluble phosphate salts orsulfate salts, enzymes, hormones, proteins, antibiotics, pyrogens,viruses, polysaccharides, colloids, surfactants, pesticides or otherinorganic substances, the separation is based on the utilization ofgravity, centrifugal force, pressure or vacuum. Exemplary processes are,inter alia: decanting, elutriation, sieving, wind-classification,classification, filtration, dialysis, sedimentation, microfiltration,ultrafiltration, flotation, foam fractionation, sink-swim separation,clarification, centrifugation or separation. Membrane separationprocesses, such as microfiltration or ultrafiltration, operated by apressure difference between feed side and permeate side are summed up asmembrane filtration. The processes differ, for example, by theirseparation limits. Thus, in the case of ultrafiltration, the cut-offlimit is not based on the particle size, for example as in the case ofmicrofiltration, but on the molar mass which is in the range from about10³ to 2×10⁶ Da. In ultrafiltration, in addition to the filtrate(permeate), what is known as the concentrate (retentate) is alsoproduced.

To separate off solid substances, or enrich or deplete dissolvedmedium-molecular-weight and high-molecular-weight substances,advantageously asymmetrically structured porous membranes are used.

The membranes used according to the invention can, in an advantageousvariant, be made up of a separation layer, which effects the actualseparation, and a one layer or multilayered supporting layer which bearsthe separation layer and has coarser pores than the separation layer.The separation layers and the supporting layer can consist of organic orinorganic polymers, ceramic, metal or carbon and must be stable in thereaction medium and at the process temperature. Examples of these arelisted in table 1, but are not limiting for the present invention:

The membranes can be used in the form of flexible tubing, tubes,capillaries, hollow fibers or flat membranes in flat, tubular,multi-channel element, capillary or coiled modules which are known perse.

The optimum transmembrane pressures between retentate and permeate areessentially from 1 to 40 bar, depending on the diameter of the membranepores or the cut-off limit (expressed in molecular weight units), themechanical stability of the membrane and the type of membrane. Highertransmembrane pressures generally lead to higher permeate fluxes. In thecase in which the feed (solution to be treated) is fed at an excessivepressure, the transmembrane pressure can be adapted by increasing thepermeate pressure.

The operating temperature is dependent on the product stability andmembrane stability. It is from about 20 to 90° C., preferably from about40 to 70° C. Higher temperatures lead to higher permeate fluxes.Membranes according to table 2 can be used, for example, but these arenot limiting for the present invention.

Cell separation, according to the invention, can advantageously also beperformed by a special type of membrane filtration, that is to saydiafiltration. Diafiltration can take place batchwise by passing thesolution containing polyvalent salts of D-pantothenic acid via a circuitcomprising a vessel, a pump and one or more membrane modules and settingthe pressures in the membrane modules in such a manner that permeate isproduced. Continuously, or at certain times, water or an aqueoussolution is added which does not contain the product to be removed, orcontains it at a lower concentration than at the timepoint of additionto the separation circuit. According to the invention, the aqueoussolution can contain salts of polyvalent cations, for example calcium ormagnesium halides or combinations thereof, preferably calcium chlorideand/or magnesium chloride.

The cell removal by means of diafiltration can according to theinvention also be performed continuously, with preferably a plurality ofmembrane modules being connected in series, or in each case one or moremembrane-module-containing pump circuits being connected in series.Upstream of, between or downstream of the membrane modules or pumpcircuits, water or an aqueous solution which does not contain theproduct to be removed or only contains it at lower concentration than atthe feed site can be added, in which case, as in the batch variant, theaqueous solution can contain salts of polyvalent cations, for examplecalcium or magnesium halides or combinations thereof, preferably calciumchloride and/or magnesium chloride.

According to the invention the ultrafiltration or diafiltration can becarried out directly using the fermentation output, or after a treatmentof the fermentation output, for example by centrifugation, decanting orsimilar procedure.

If according to the invention cell mass or components precipitated outin the solution are removed, the addition of salts containing polyvalentcations according to step b) of the inventive process can take placeduring, or after, the ultrafiltration or diafiltration. In variants ofthe inventive process, polyvalent cations added are, for example,calcium and/or magnesium chloride, nitrate, hydroxide, formate, acetate,propionate, glycinate and/or lactate. In this case the polyvalentcation, for example Ca²⁺, can be fed at a concentration of 0.05-50 molof Ca²⁺/mol of D-pantothenate, preferably 0.2-2 mol of Ca²⁺/mol ofD-pantothenate.

In step c) of the inventive process, the solution containing polyvalentsalts of D-pantothenic acid is then worked up by nanofiltration, thepolyvalent salts of D-pantothenic acid being enriched and simultaneouslyunwanted monovalent ions, preferably monovalent cations, for exampleammonium, sodium or potassium ions, are depleted. In the inventiveprocess, the content of monovalent cations, preferably ammonium,potassium and/or sodium ions, is reduced to a concentration of ≦5 g/kgof solution.

The present invention comprises all commercially availablenanofiltration systems. The separation is advantageously performed onasymmetrically structured porous membranes. In a preferred variant ofthe present process, membranes are used for this which are made up of aseparation layer which carries out the actual separation, and a single-or multi-layered support layer which bears the separation layer and hascoarser pores than the separation layer. The separation layers and thesupport layer can consist of organic polymers, ceramic, metal or carbonand must be stable in the reaction medium and at the processtemperature. Preferred materials for the separation layer arepolyamides, polyimides, or polypiperazines. The separation layers canalso have a positive or negative surface charge. An example of ananionically functionalized nanofiltration membrane is the membrane DESAL5 DK, but the present invention is not limited to the exclusive use ofthis membrane.

The membranes can be used in the form of flexible tubings, capillaries,hollow fibers or flat membranes, and in flat, tubular,multi-channel-element, capillary or coil modules which are known per se.

In advantageous variants of the inventive process, in the nanofiltrationin step c), a pressure difference over the membrane in the range of5-100 bar, preferably 20-80 bar, and particularly preferably 40-70 bar,is built up.

The process temperature is advantageously from 20 to 80° C., preferablyfrom 30 to 60° C. In addition, the nanofiltration can be carried out ina manner known to those skilled in the art continuously or batchwise inone or more steps.

In a preferred variant, in each case before one or more nanofiltrationstep(s), a salt containing polyvalent cations is added in solid form orin aqueous solution.

According to the invention, the polyvalent cations are added as calciumand/or magnesium chloride, nitrate, hydroxide, formate, acetate,propionate, glycinate and/or lactate. In this case, as polyvalentcation, Ca²⁺ can be fed at a concentration of 0.05-50 mol of Ca²⁺/mol ofD-pantothenate, preferably 0.2-2 mol of Ca²⁺/mol of D-pantothenate(based on the state after mixing).

In the inventive process, salts containing polyvalent cations are addedin solid form or aqueous solution during, preferably at the end of, orafter, the fermentation in step a) or during or after the cellseparation.

According to the invention, moreover, adding salts containing polyvalentcations during the nanofiltration step can be advantageous. In addition,an aqueous solution containing polyvalent cations can be suppliedcontinuously.

In a further variant of the present process, it is conceivable that, inone or more process steps upstream of the nanofiltration solutions ofdiffering product concentration can arise. Said solutions can be furtherprocessed by a nanofiltration in such a manner that said solutions aresupplied in sequential nanofiltration steps in the order of ascendingproduct concentrations.

According to the invention, as a result of the above-described inventiveprocess, primarily the polyvalent salts of pantothenic acid are enrichedin the retentate of the nanofiltration. In the permeate solution,principally the monovalent ions are enriched, when an anionicallyfunctionalized nanofiltration membrane is used, the monovalent cationsare enriched. The content of monovalent cations, preferably ammonium,potassium and/or sodium ions, in the retentate can be reduced in thiscase to a concentration of ≦5 g/kg of solution. According to theinvention the permeate of the nanofiltration or a part thereof can berecirculated to the fermentation in step a) of the inventive process.This recirculation of the permeate or parts thereof can be performedcontinuously. The above-described process steps carried out in additionto the nanofiltration serve for preconcentration or furtherconcentration of D-pantothenate in the form of polyvalent salts.

A further advantage of the inventively used nanofiltration is that thereduction of the monovalent cations (in the retentate solution) can beaccompanied simultaneously with a volume reduction of the retentate. Theworkup of the D-pantothenate-containing fermentation solution viananofiltration can thus be used according to the invention asion-exchange and concentration process for producing D-pantothenate.

This leads advantageously to a simplification, and simultaneouslyincreased efficiency, of the subsequent process steps. For example, theenergy consumption in drying can be significantly reduced on account ofthe concentration.

In a preferred embodiment of the present invention, the fermentationsolution is freed from the cell mass by centrifugation and/ordecantation and/or ultrafiltration. After adding from 0.05 to 50 mol(Ca²⁺) ions/mol of pantothenate ion, preferably 0.2-2 mol Ca²⁺ ions/molof pantothenate ion, which are preferably charged in the form of adilute solution having 0.01-10 mol of Ca²⁺/1, the resultant solution isintroduced into a nanofiltration module. The pressure difference acrossthe membrane is in the range of about 5-100 bar, preferably about 20-80bar, particularly preferably about 40-70 bar. Before or during thenanofiltration in this case, a Ca²⁺-ion-containing aqueous solution canbe added to the solution flowing across the membrane on the feed side.The retentate has a volume of 30-200% of the starting solution. Inaddition, about 5-99%, preferably 30-80%, of the monovalent cationspresent are removed.

The retentate preferably containing calcium D-pantothenate, magnesiumD-pantothenate or a mixture thereof is then subjected to a drying and/orformulation. The drying and/or formulation of the calcium- and/ormagnesium-D-pantothenate-containing solution is performed by methodsknown per se, for example spray drying, spray granulation, fluidized-beddrying, fluidized-bed granulation, drum drying, or spin-flash drying(Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 1999,electronic release, chapter “Drying of Solid Materials”). The gas inlettemperature in convection drying is in the range 100-280° C., preferably120-210° C. The gas outlet temperature is 50-180° C., preferably 60-150°C. To establish a desired particle size distribution and the associatedproduct properties, fine particles can be separated off andrecirculated. In addition, coarse material can be ground in a mill andlikewise then recirculated.

The inventive process has the advantages that unwanted cations areefficiently and virtually completely removed and at the same time areduction in volume takes place which makes the subsequent processsteps, in particular drying and/or formulation, simplified or moreefficient. In addition, no product decomposition, or only extremely lowproduct decomposition, takes place, with simultaneously high productyield. By feeding salt solutions of polyvalent cations during, or at theend of, fermentation, or during, or at the end of, an ultrafiltration ordiafiltration, or during the nanofiltration step and/or by recirculatingthe permeate to the fermentation solution, the yields of D-pantothenatein the form of polyvalent, preferably divalent, ions, such as calcium ormagnesium, are further increased.

In the above-described process, in addition, according to the inventionreducing complex workup steps is advantageous, in particular omittingthe use of organic solvents, with simultaneous provision of a desiredproduct of good biological value. In addition, according to theinvention the amount of wastewater produced is substantially reduced.This thus results in further savings in complex treatment and disposalplants. Thus the inventive process is advantageously distinguished inthat it is simpler, less susceptible to faults, less time-consuming,significantly less expensive and thus more economical, than conventionalprocesses.

However, this does not exclude the inventive process from being able tobe varied. The previously described inventive process can besupplemented by one or more of the following process steps, each ofwhich is familiar to those skilled in the art. In this case, allconceivable combinations of the following process steps with the processsteps known to date are included according to the invention.

Thus the solutions resulting from the inventive process can bedisinfected, for example by heating (sterilization) or other methods,for example pasteurization or sterile filtration.

In further variants of the inventive process, before drying and/orformulation of the retentate, at least one, or combinations, of thefollowing steps can be carried out, comprising lysis and/orsterilization of the biomass and/or separation of the biomass from thefermentation solution and/or addition of further additives and/orconcentration of the fermentation solution, preferably by removal ofwater.

The present invention thus also relates to a process in which the lysisand/or sterilization of the biomass is carried out while still in thefermentation solution, or not until after the biomass is separated offfrom the fermentation solution. This can be performed, for example, by atemperature treatment, preferably at 80-200° C., and/or an acidtreatment, preferably with sulfuric acid or hydrochloric acid and/orenzymatically, preferably with lysozyme.

It is also conceivable for the cell mass present to be removed directlyvia the nanofiltration, that is to say simultaneously with the exchangeof monovalent cations against polyvalent cations.

The solution resulting from the workup via nanofiltration can beconcentrated before the drying and/or formulation via a suitableevaporator, e.g. falling-film evaporator, thin-film evaporator, orrotary evaporator. Such evaporators are manufactured, for example, bythe company GIG (4800 Affnang Puchheim, Austria), GEA Canzler (52303Düren, Germany), Diessel (31103 Hildesheim, Germany) and Pitton (35274Kirchhain, Germany).

To improve the color properties of the end product, an additionalfiltration step can be carried out, in which a little activated carbonis added to the solutions obtained during the process and thissuspension is then filtered. Or, the solutions obtained duringfermentation can be passed through a small activated-carbon bed. Theamounts of activated carbon used required for this are in the range of afew percent by weight of the solution and are within the knowledge andjudgement of a person skilled in the art.

These filtrations can be simplified by adding a commerciallyconventional flocculant (e.g. Sedipur CF 902 or Sedipur CL 930 from BASFAG, Ludwigshafen) to the respective solution before filtration.

In an advantageous embodiment of the present invention, the fermentationoutput (fermentation broth) is sterilized by heating and then freed fromthe cell mass by centrifugation, filtration, ultrafiltration ordecantation. After addition of 50-1 000 mg/kg, preferably 100-200 mg/kg,of a commercially conventional flocculant, based on the fermentationoutput, the suspension is filtered through a short bed of activatedcarbon and sand to obtained a biomass-free solution having a highD-pantothenic acid content. This treated solution is then treated bynanofiltration.

The subsequent drying of this solution can be performed, for example, byspray drying. This can be performed in cocurrent, countercurrent ormixed-stream flow. For atomization, all known atomizers can be used, inparticular centrifugal atomizers (atomizer disk), single-fluid nozzle ortwo-fluid nozzle. Preferred drying temperature conditions are 150-250°C. tower inlet temperature and 70-130° C. tower outlet temperature.However, drying can also be performed at a higher or lower temperaturelevel. To achieve very low residual moisture, a further drying step in afluidized bed can be provided downstream.

The spray drying may also be carried out in an FSD or SBD dryer (FSD:fluidized spray dryer; SBD: spray bed dryer), as are constructed by thecompany Niro (Copenhagen, Denmark) and APV-Anhydro (Copenhagen,Denmark), which are a combination of spray-dryer and fluidized bed.

In spray drying, a flow aid can be added. As a result the deposits onthe dryer wall can be reduced and the flow behavior particularly in thecase of fine-grained powders, can be improved. Flow aids which can beused are, in particular, silicates, stearates, phosphates and cornstarch.

In principle, drying can also take place in a spray fluidized bed, inwhich case this can be operated not only continuously but alsobatchwise. The solution can be sprayed in not only from the top(top-spray), from the bottom (bottom-spray), but also from the side(side-spray).

The present invention further relates to a composition for the use asanimal feed additive and/or animal feed supplement, in which it can beprepared by

-   -   a) using at least one D-pantothenic-acid-producing organism, the        pantothenic acid (pan) and/or isoleucine/valine (ilv)        biosynthesis of which is deregulated and which forms at least 2        g/l of salts of D-pantothenic acid by fermentation in a culture        medium, 0-20 g/l, preferably 0 g/l, of free β-alanine and/or        β-alanine salt being fed to the culture medium,    -   b) feeding salts containing polyvalent cations to the        D-pantothenate formed, polyvalent salts of D-pantothenic acid        being produced,    -   c) treating the D-pantothenate-containing fermentation solution        by nanofiltration, the polyvalent salts of D-pantothenic acid        being enriched,    -   d) subjecting the nanofiltration retentate containing polyvalent        salts of D-pantothenic acid to a drying and/or formulation.

In a variant of the present invention, a composition is comprised whichcan be prepared by, before the nanofiltration in step c), carrying out aremoval of cell mass or components precipitated out in the solution,preferably by membrane filtration, particularly preferably byultrafiltration, and very particularly preferably by diafiltration. Thepresent invention further relates to a composition which can be preparedby feeding salts (in solid form or as aqueous solution) containingpolyvalent cations during, or after, the removal of cell mass orcomponents precipitated out in solution. According to the inventionthese salts, in a further variant, can also be supplied during thenanofiltration.

According to the invention the composition is further distinguished inthat it contains salts of D-pantothenic acid in a concentration of atleast 1-100% by weight, preferably 20-100% by weight and particularlypreferably at least 50% by weight. The present invention relates to acomposition which contains salts of D-pantothenic acid in the form ofdivalent cations, preferably calcium D-pantothenate and/or magnesiumD-pantothenate. According to the invention preference is given to acomposition which is distinguished in that the content of salts ofD-pantothenic acid in the form of monovalent cations is ≦5 g/kg.

According to the invention, owing to the above-described process, acalcium D-pantothenate or magnesium D-pantothenate is obtained whichsatisfies the requirements of a food additive. These requirements are,for example, a relatively high content of D-pantothenate, and a highcompatibility for the target organism, and also a biological value inthe sense of the “vitamin activity” of the inventive product.

The present invention is described in more detail by the examples below,which are not limiting for the invention, however:

EXAMPLE 1

In a laboratory fermenter equipped with agitator and gas-introductiondevice of 14 l capacity, aqueous fermentation medium of the followingcomposition is charged:

Concentration Starting material [g/l] Yeast extract 5 Soybean meal 40Sodium glutamate · H₂O 5 Ammonium sulfate 8 KH₂PO₄ 5 K₂HPO₄ 10 NaH₂PO₄ ·2 H₂O 6.15 NaH₂PO₄ · 2 H₂O 12

After sterilization, the following sterile media components were addedin addition:

Concentration Starting material [g/l] Glucose · H₂O 20 Calcium sulfate0.1 Magnesium sulfate 1 Sodium citrate 1 FeSO₄ · 7 H₂O 0.01 Trace saltsolution 1 ml

The trace salt solution had the following composition: 0.15 g ofNa₂MoO₄.2H₂O, 2.5 g of H₃BO₃, 0.7 g of CoCl₂.6H₂O, 0.25 g of CuSO₄.5H₂₀,1.6 g of MnCl₂.4H₂O, 0.3 g of ZnSO₄.7H₂O were made up to 1 l with water.The trace salt solution was added through sterile filtration. Theinitial liquid volume was 5 l. The contents listed above are based onthis value.

To this solution were added 100 ml of inoculation culture (OD=10) ofBacillus subtilis PA668 and the inoculated culture was fermented at 43°C. with vigorous stirring at a gas-flow rate of 12 l/min. This strain isdescribed according to the annex of U.S. application serial No.60/262,995.

In the course of 47 h, 2.1 l of a sterile aqueous solution were added.The composition was:

Concentration Starting material [g/l] Glucose 800 Calcium chloride 0.6Sodium glutamate · H₂O 5 Sodium citrate 2 FeSO₄ · 7 H₂O 0.2 Trace saltsolution 6 ml

During the fermentation, the pH was kept at 7.2 by adding 25% strengthammonia solution or 20% strength phosphoric acid. Ammonia simultaneouslyacts as nitrogen source for the fermentation. The speed of rotation ofthe agitator element was controlled to keep the dissolved oxygen contentat 30% of the saturation value. After terminating the addition of thecarbon source, the fermentation was continued until the dissolved oxygencontent (pO₂) had reached a value of 95% of the saturation value. Theconcentration of D-pantothenate at termination after 48 h was 22.8 g/l.

Similarly, fermentation broths can also be produced which giveβ-alanine-supply-free pantothenic acid titers greater than 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, and >90 g/l.

EXAMPLE 2

7 000 ml of the fermentation output produced according to example 1 weresubjected to ultracentrifugation, a ceramic monochannel tubular module(from Atech, Gladbeck, Germany) being used. In this case firstly amembrane having a pore width of 20 kD (10 nm) and secondly a membranehaving a pore width of 50 nm were used.

The temperature in the experiments was 40° C., the overflow speed was 4m/s and the transmembrane pressure(TMP=[p(feed)+p(retentate)]/2−p(permeate)), unless stated otherwise, was1 bar.

In FIG. 1 the transmembrane fluxes (permeate fluxes) are plotted as afunction of the concentration factor MK (MK(t)=m_(feed)/m_(retentate)(t)).

It becomes apparent that the membrane having the lower pore width (20kD) exhibits markedly higher fluxes than the membrane having the greaterpore width (50 nm).

EXAMPLE 3

7 000 ml of the fermentation output, produced as under example 1, weresubjected to ultrafiltration [sic] similar to example 2, the membraneused having a pore width of 20 kD.

FIG. 2 shows that the concentration using fermentation output which hadalready been centrifuged is markedly higher than in example 2.

EXAMPLE 4

1 000 ml of an aqueous solution containing calcium pantothenate(according to table 3, column retentate feed) were placed in an agitatorpressure cell having a maximum operating capacity of approximately 1.5l. The “feed” pressure is generated in this cell by nitrogenoverpressure and the membrane overflow was ensured by agitation using ananchor agitator driven by a magnetic coupling.

The nanofiltration membrane DESAL 5 DK, obtained from OsmonicsDeutschland GmbH in Moers, was used.

Before, during and after the experiments with the abovementionedsolution, to test the membrane integrity, rejection tests with MgSO₄solution (2 000 ppm by weight) were carried out.

The rejection rate R_(i) is listed in the two right-hand columns oftable 3. Here the rejection rate is defined as follows: R_(i)=1−c_(i,permeate)/c_(i,retentate); where R_(i)=rejection rate forcomponent i, c_(i,permeate)=concentration of component i in thepermeate, c_(i,retentate)=concentration of component i in the retentate.

The concentrations mean the concentrations instantaneously establishedin the non-steady state experiment at a defined timepoint, but not theconcentration in the fractions resulting after the end of theexperiment. The rejection rate R_(i), in the ideal case, isconcentration-independent, which was also used as a basis in calculatingthe reported values from the concentrations in the fractions.

From table 3 it follows that Ca ²⁺ and pantothenate have rejection ratesof 84% and 99%, respectively, that is to say are present in theretentate.

EXAMPLE 5

In the workup of an aqueous solution of Na pantothenate (0.2 mol/l),under similar conditions to example 4, a concentration by nanofiltrationwas carried out. Table 4 shows that the rejection rate of pantothenateis 80%.

EXAMPLE 6

Concentrating an equimolar solution of NaCl/CaCl₂ under similarconditions to example 4 is summarized in table 5. Here it can be seenthat the rejection rate of the membrane for Ca²⁺ of 41% or 42% isrelatively low compared with the high rejection rate of calcium incombination with pantothenate (example 5).

Legend to the Figures and Tables

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Graphical presentation of transmembrane fluxes (permeate fluxes)of fermentation output during ultrafiltration as a function ofconcentration factor MK using membranes having a pore width of 50 nm and20 kD.

FIG. 2: Graphical presentation of transmembrane fluxes (permeate fluxes)of a centrifuged fermentation output during ultrafiltration as afunction of concentration factor MK using a membrane having a pore widthof 20 kD.

Table 1:Outline of asymmetrically structured membranes for separatingoff cell mass or components precipitated out in solution.

Table 2: Outline of membranes and their properties for separating offcell mass or components precipitated out in solution.

Table 3: Outline of the analytical values of nanofiltration, especiallywith respect to the rejection rate of calcium ions and pantothenate, inan aqueous solution containing 0.1 mol/kg of Ca pantothenate and 0.2mol/kg of NaCl.

Table 4: Outline of the analytical values of nanofiltration, especiallywith respect to the rejection rate of calcium ions and pantothenate, inan aqueous solution containing 0.2 mol/l of Na pantothenate and 0.1mol/l of CaCl₂.

Table 5: Outline of the analytical values of nanofiltration, especiallywith respect to the rejection rate of calcium ions, in an aqueoussolution containing equimolar amounts of NaCl and CaCl₂.

1. A process for preparing D-pantothenic acid and/or salts thereof,which comprises a) fermenting at least one D-pantothenic-acid-producingbacterium from the Bacillaceae family, the pantothenic acid (pan) andoptionally the isoleucine/valine (ilv) biosynthesis of which isderegulated and which forms at least 2 g/l of salts of D-pantothenicacid by fermentation in a culture medium, b) salts containing polyvalentcations being fed to the D-pantothenate formed, polyvalent salts ofD-pantothenic acid being formed, c) the solution containing polyvalentsalts of D-pantothenic acid being worked up by nanofiltration, thepolyvalent salts of D-pantothenic acid being enriched and d) thenanofiltration retentate containing polyvalent salts of D-pantothenicacid being subjected to drying and/or formulation, wherein freeβ-alanine and/or β-alanine salt is not fed to the culture medium, andwherein deregulated panthothenic acid biosynthesis is achieved byoverexpressing one or more genes in the pan and optionally in the ilvsynthesis pathways, wherein said overexpressed genes include at leastthe panD gene.
 2. A process as claimed in claim 1, wherein the bacteriumis of the genus Bacillus.
 3. A process as claimed in claim 1, wherein,in step a), a content of D-pantothenic acid and/or salts thereof of atleast 10 g/l of culture medium is formed.
 4. A process as claimed inclaim 1, wherein cell mass or components precipitated out in thesolution is/are separated off before the nanofiltration in step c).
 5. Aprocess as claimed in claim 4, wherein the separation is carried out bydecanting or membrane filtration.
 6. A process as claimed in claim 5,wherein the membrane filtration is carried out as diafiltration.
 7. Aprocess as claimed in claim 5, wherein salts containing polyvalentcations are added in solid form or aqueous solution during, at the endof, or after, the fermentation in step a) or during, or after, amembrane filtration of a D-pantothenate-containing solution.
 8. Aprocess as claimed in claim 7, wherein salts containing polyvalentcations are added in solid form or aqueous solution during ananofiltration.
 9. A process as claimed in claim 7, wherein an aqueoussolution containing polyvalent cations is fed continuously.
 10. Aprocess as claimed in claim 7, wherein the polyvalent cations are addedas calcium and/or magnesium chloride, nitrate, hydroxide, formate,acetate, propionate, glycinate and/or lactate.
 11. A process as claimedin claim 7, wherein the polyvalent cation fed is Ca²⁺ at a concentrationof 0.05-50 mol of Ca²⁺/mol of D-pantothenate.
 12. A process as claimedin claim 1, wherein, in the nanofiltration in step c), a pressuredifference across the membrane in the range of 5-100 bar is built up.13. A process as claimed in claim 1, wherein the nanofiltration in stepc) reduces the content of monovalent cations to a concentration of ≦5g/kg of solution.
 14. A process as claimed in claim 1, wherein thepermeate from step c) or a part thereof is recirculated to thefermentation in step a).
 15. A process as claimed in claim 1, whereinthe permeate or parts thereof are recirculated continuously.
 16. Aprocess as claimed in claim 1, wherein the retentate from c) is asuspension containing polyvalent salts of D-pantothenic acid.